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Plaunotol from Croton stellatopilosus Ohba inhibited cell

growth and induced apoptosis in human cancer cell lines

Journal: Songklanakarin Journal of Science and Technology

Manuscript ID SJST-2017-0436.R2

Manuscript Type: Original Article

Date Submitted by the Author: 05-Mar-2018

Complete List of Authors: Premprasert, Charoenwong; Pharmacognosy and Pharmaceutical Botany Yuenyongsawad, Supreeya; Fac. of Pharm. Sci., Prince of Songkla University, Tewtrakul, Supinya; Fac. of Pharmaceutical Sciences, Prince of Songkla University, Pharmacognosy and Pharmaceutical Botany Wungsintaweekul, Juraithip; Prince of Songkla University, Pharmacognosy

and Pharmaceutical Botany

Keyword: plaunotol, anti-proliferative activity, MTT assay, apoptosis, human cancer cell lines

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Songklanakarin Journal of Science and Technology SJST-2017-0436.R2 Wungsintaweekul

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1

Original article

Plaunotol from Croton stellatopilosus Ohba inhibited cell growth and induced

apoptosis in human cancer cell lines

Charoenwong Premprasert, Supreeya Yoenyongsawad, Supinya Tewtrakul,

and Juraithip Wungsintaweekul*

Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical

Sciences, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand

*Corresponding author.

Email address: [email protected]

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Abstract

Plaunotol, an acyclic diterpene alcohol, was evaluated for an anti-proliferative

activity in human cancer cell lines including HeLa, HT-29, MCF-7 and KB cells. After

treatment cells with plaunotol, cell viability was determined using MTT assay. The

results showed that plaunotol inhibited the growth of human cancer cell lines with IC50

of 65.47 ± 6.39 µM (HeLa), 72.92 ± 5.73 µM (HT-29), 80.90 ± 3.48 µM (KB) and

62.25 ± 9.15 µM (MCF-7), respectively. For apoptotic detection, treated cells with

plaunotol were stained with Annexin-V/7-AAD reagent. The results indicated that

plaunotol induced cell death or apoptosis. The transcription profile of apoptotic-

associated genes including TNF-α, BCL-2, BAX and BAK genes was determined using

qRT-PCR technique and the expression level was calculated as the relative quantitation

(RQ). The ratio between RQ of BCL-2 and BAX suggested that plaunotol significantly

induced apoptosis in HeLa, MCF-7 and HT-29 cell lines, in particular.

Keywords: plaunotol, anti-proliferative activity, MTT assay, apoptosis, human cancer

cell lines

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1. Introduction

Plaunotol (Fig. 1) is an acyclic diterpene alcohol derived from geranylgeranyl

diphosphate (GGPP). It has been isolated since 1978 and was only found in Croton

stellatopilosus Ohba or plaunoi [Thai name] (Ogiso et al., 1978). Plaunotol was claimed

to have anti-peptic ulcer ability and it has been successfully registered with the World

Health organization (WHO) (Ogiso et al., 1985). It was further processed into a soft

gelatin capsule under the trade name Kelnac®

by Sankyo Daiichi, Japan. Recently, the

partially purified plaunotol extract was reported to be potentially safe after acute and

chronic oral toxicity test in animal model (Chaotham, Chivapat, Chaikitwattana, & De-

Eknamkul, 2013).

Plaunotol has gastro-protective effect that mediates several mechanisms such

as releasing prostaglandins (PGE2 and PGI2), secretin and inhibiting neutrophil

activation (Ushiyama et al., 1987; Shiratori, Watanabe, & Takeuchi, 1993; Murakami et

al., 1999). Considering Croton species, some diterpenoids have been reported for

anticancer and antitumor activities such as trans-dehydrocrotonin and trans-crotonin

from C. cajucara (Grynberg et al., 1999), neocrotocembranal from the stem bark of C.

oblongifolius (Roengsumran et al., 1999), ent-15-oxo-kaur-16-en-18-oic acid from the

bark of C. argyrophylloides (Santos et al., 2009). Nowadays, several secondary

metabolites with interesting biological activities serve as leading compounds in the

development of drugs such as paclitaxel and docetaxel. Both drugs are strong anticancer

agents and are already being used in the treatment of cancer. Interestingly, other

isoprenyl molecules such as farnesol (Joo, Liao, Collins, Grissom, & Jetten, 2007; Park

et al., 2014), geranylgeraniol (GGOH) (Ohizumi et al., 1995; Yoshikawa et al., 2009),

geranylgeranoic (Nakamura et al., 1996; Shidoji, Nakamura, Moriwaki, & Muto, 1997;

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Shidoji et al., 2006) and geranylgeranylacetone (Yoshikawa et al., 2010; Jo et al.,

2016), have been reported to inhibit cancer cell growth via apoptosis induction.

Plaunotol has been identified as a cytoprotective anti-peptic ulcer agent. Up to

date, plaunotol has been reported to possess antitumor activity against gastric cancer

cell lines (MKN-45, MKN-74 and AZ-521) by apoptosis. It inhibited the growth of

gastric cancer cells and induced caspase activation including caspase-3, -8, and -9

(Yamada et al., 2007). Moreover, plaunotol was reported to exhibit cytotoxic effect

against DLD1 human colon cancer cell line (Yoshikawa et al., 2009).

Since the discovery of signaling pathways in human cancer cells, these

pathways have shown to be connected with the network of regulation at the molecular

level of cell growth and cell division. Apoptosis is a biological process in multi-cellular

organism that plays an important role in animal development and homeostasis (Fuchs &

Steller, 2011). During apoptosis, the cell initially defined by its biochemical

characteristic changes, including cell shrinkage, blebbing of the plasma membrane,

nuclear morphology (which includes the chromatin condensation and fragmentation,

and releasing the apoptotic mediators) (Prayong, Weerapreeyakul, & Barusrux, 2007).

Cell cycle is regulated by a complex sequence of signaling pathways by which a cell

grows, duplicates its DNA and divides (Cooper, 2000). Nevertheless, in cancer cells,

cell cycle process is a failure and results in an uncontrolled cell proliferation. For this

reason, a better understanding of cancer biology and cancer genetics is necessary for

cancer research. Among these researches, many reports were focused on the anti-

proliferative effect of cancer cells, its mechanisms and the realization that cell cycle

progression, apoptosis and regulatory genes involve apoptosis and cell cycle.

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Based on the above mentioned rationale, plaunotol may exert anticancer effects

in human cancer cell lines. Therefore, the present study was undertaken to assess the

effect of plaunotol on the growth of cancer cells and also its mechanism was

investigated via apoptosis. The study reported the investigation of molecular mechanism

of plaunotol on anti-proliferative effect, induction of apoptosis and accumulation of

cells in cell cycle phase. In addition, the transcription analysis of associated-apoptotic

mediators on four cancer cell lines including HeLa, HT-29, MCF-7 and KB cells was

also investigated.

2. Materials and Methods

2.1 Isolation of plaunotol

Plaunotol was extracted from C. stellatopilosus leaves using silica gel column

chromatography as described in Premprasert et al. (2013). The structure was confirmed

by spectroscopic methods [1H-,

13C-NMR and MS].

Plaunotol : a pale yellow oil; IR νmax cm-1

3300 (O-H), 1665 (C=C), 1440 (C-

H), 1380, 1000; 1H NMR (CDCl3, 500 MHz) δ: 1.60 (3H, s, H-19), 1.60 (3H, s, H-20),

1.64 (3H, s, H-16), 1.64 (3H, s, H-17), 1.95 (2H, m, H-13), 2.02 (2H, m, H-12), 2.02

(2H, m, H-4), 2.10 (2H, m, H-8), 2.16 (2H, m, H-5), 4.05 (2H, s, H-18), 4.07 (2H, d, J =

7.1 Hz, H-1), 5.06 (1H, m, H-10), 5.09 (1H, m, H-14), 5.22 (1H, m, H-6); 13

C NMR

(CDCl3, 125 MHz) δ: 123.9 (C-10), 124.0 (C-14), 124.2 (C-2), 127.4 (C-6), 131.3 (C-

11), 135.3 (C-15), 138.8 (C-7), 138.9 (C-3), 15.9 (C-20), 16.4 (C-17), 17.6 (C-19), 25.6

(C-16), 25.8 (C-5), 26.6 (C-13), 26.7 (C-9), 34.8 (C-8), 39.2 (C-4), 39.6 (C-12), 58.9

(C-18), 59.8 (C-1); EI-MS at m/z 306.255 (M+).

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2.2 Chemicals

All chemicals used in this study were of analytical grade. Dulbecco’s Modified

Eagles’s Medium (DMEM), trypan blue, trypsin–EDTA, fetal bovine serum (FBS) and

MTT; 3-(4, 5-dimethyl-2-thiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide was from

Gibco®

BRL, CA, USA. The antibiotics [Penicillin G (100 U/mL) plus streptomycin

(100 µg/mL)] was supplied by Invitrogen

, CA, USA. The Total RNA mini kit used for

total RNA isolation was from Geneaid®

, New Taipei City, Taiwan. TaKaRa One Step

SYBR®

PrimeScript™ RT-PCR Kit II was purchased from Takara Bio Inc., Japan.

Primers used for qRT-PCR were designed from the Gene Bank information of Homo

sapiens (http://ncbi.nlm.nih.gov). Assay kits; Muse™ Annexin-V εt Dead cell reagent

and Muse™ cell cycle reagents were purchased form Merck, Darmstadt, Germany.

2.3 Cell lines

Cells, including human breast carcinoma cell line (MCF-7; CLS No. 300273),

human cervical carcinoma cell line (KB, CLS No. 300446), human cervix

adenocarcinoma (HeLa, CLS No. 300194), and human colon adenocarcinoma (HT-29,

CLS No. 300215) were obtained from the Cell Line Service, Heidelberg, Germany. The

human gingival fibroblast (HGF) cell line was kindly provided by the Faculty of

Dentistry, Prince of Songkla University. Cells were maintained in DMEM

supplemented with 10% FBS and 2% antibiotics (penicillin and streptomycin) at 37°C

in a humidified atmosphere containing 5% CO2. The mono-layered cells were sub-

cultured weekly with 0.25% trypsin-EDTA when they reached 80% confluence.

2.4 Cell viability assay

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Cell viability was evaluated by the MTT assay with some modifications

(Mosmann, 1983). The cells were washed twice with PBS briefly then harvested using

trypsin-EDTA. The harvested cells (1×103 cells per well) were each placed in a well of

96-well plate. These cells were attached to a plate after 24 h incubation at 37°C in an

atmosphere of 5% CO2. Aliquots of medium containing different concentrations of

plaunotol (3, 10, 30 and 100 µM) were added and the cell cultures incubated for 48 h.

This experiment established that the use of DMSO concentrations (0.2 % v/v) in the cell

cultures caused no cell damage. After the incubation period, the culture medium was

removed and washed twice with PBS. Then 100 µL of MTT reagent (5 mg/mL in PBS)

was added to each well and the cells were incubated for 3 h. The cell viability was

assessed by the ability of metabolically active cells to reduce tetrazolium salt to

formazan crystal. The obtained formazan crystals were dissolved in acidic condition

(100 µL of 0.01 N HCl in isopropyl alcohol). The absorbance of the samples was

analyzed with a microplate reader (DTX 880 multimode detector, Backman Coulter Inc,

Austria) using a test wavelength of 570 nm. The correlation between concentration and

% inhibition was interpreted for IC50 value in micromolar unit.

2.5 Apoptosis detection

To evaluate the apoptotic activity of plaunotol, Annexin-V and 7-AAD double

staining was carried out, as described by Vermes, Haanen, Steffens-Nakken, H.&

Reutelingsperger (1995). Human cancer cell lines: HeLa, HT-29, KB and MCF-7 were

separately seeded briefly at 5×105 cells per wells and treated with 75 µM and 150 µM of

plaunotol for 48 h. Paclitaxel at concentration of 1 µM was used as positive control.

After treatment, cells were washed twice with PBS and harvested with 0.25% trypsin-

EDTA, followed by centrifugation at 500×g for 5 min. Then, cell pellet was re-

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suspended in 100 µl of DMEM media containing 1% FBS followed by the addition of

100 µl of Muse™ Annexin V εt Dead cell reagent. The solution was incubated for 20

min at room temperature in dark condition. Finally, the stained cells were analyzed by

flow cytometer using Muse™ cell analyzer. The data was analyzed using Muse™ 1.4

software and the data was shown as four-quadrant dot plot. The statistic on four cell

populations was obtained and the populations in each quadrant predicted the apoptosis

in live, early apoptotic, late apoptotic and dead, respectively.

2.6 Cell cycle analysis

HeLa, HT-29, KB and MCF-7 cancer cells were seeded into 6-well plate at

5×105 cells/well, then cells were treated with varying doses of plaunotol (25 µM to 100

µM) and with DMSO (0.2%) as a control group. Treated cells were incubated for 48 h.

After incubation, the cells were harvested using trypsin-EDTA and centrifuged at 500×g

for 5 min. Then, the cells were washed with PBS and fixed with ice cold ethanol (70%

in water). After ethanol fixation for 3 h at -20°C, the cells were then centrifuged at

500×g for 5 min. Ethanol was removed and the fixed cell was rinsed with PBS. The cell

pellet was re-suspended in PBS (0.25 mL per 5×105 cells/well), centrifuged after which

the supernatant was discarded. The MuseTM

cell cycle kit reagent (200 µL) was added;

the cells were re-suspended and incubated for 30 min in the dark at room temperature.

The stained cells were analyzed by flow cytometer (Muse™ cell analyzer). The DNA

content was analyzed and the dot plot was recorded. The histogram of the DNA content

index was generated and analyzed the cell populations in each phase of the cycle. The

cell cycle assay exploited propiodium iodide-based staining of DNA content and

measured the percentage of cells in each cell cycle phase (G0/G1, S and G2/M).

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2.7 Primers design

The sequences of genes including TNF-α, BCL-2, BAX, BAK and GAPDH

were retrieved from GenBank Database (https://www.ncbi.nlm.nih.gov/).

Oligonucleotides with 20-24 bp and theoretical melting temperature ranging from 60 -

64 °C were designed accordingly, using Primer3 Software

(http://simgene.com/Primer3). List of primers are shown in Table 1.

2.8 Quantitative real-time PCR (qRT-PCR)

The human cancer cell lines (5×105 cells per well) in 6-well plate were treated

with plaunotol at concentrations of 50 µM and 75 µM and incubated further for 48 h at

37°C in a 5% CO2 humidified atmosphere. The cells were treated with 0.2% (v/v)

DMSO and paclitaxel (1 µM) as negative control and positive control, respectively.

After incubation, the cells were washed with PBS and treated with trypsin-EDTA. The

suspension of cells was harvested by centrifugation at 500×g for 5 min. Supernatant was

removed and the cell pellet was rinsed with PBS and stored at -80°C until use.

The total RNA was isolated from the cells using Total RNA Mini Kit (Geneaid,

Taiwan) according to the manufacturer’s protocol. The quality and quantity of total

RNA of each treated cells were evaluated by UV spectrophotometer (Thermo Scientific,

USA). The mRNA levels of genes were determined using qRT-PCR (ABI Prism®

7300)

in the presence of RNA template and One Step SYBR®

PrimeScriptTM

RT-PCR kit II

(Perfect Real Time, Takara, Japan). The qRT-PCR was carried out in a final volume of

20 µL reaction; with mixture containing 0.8 µL of each primer (forward & reward)

(Table 1), 10 µL of 2x one step RT-PCR buffer IV, 0.4 µL of ROX dye (50x), 2 µL of

RNA (20 ng) as a template and 0.8 µL of PrimeScript one step enzyme mix II, and

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adjusted to the volume with RNase free water. At optimal PCR condition, the

amplification plot and melting curve were generated. Cycle number at threshold (set at

0.2) was recorded. The GAPDH gene was used as endogenous gene. Expression level of

control treatment was used as a calibrator of each gene. The transcription profile of gene

was expressed as relative quantitative (RQ), which was calculated using the

comparative CT method when RQ = 2-∆∆C

T.

2.9 Statistical analysis

All the data represented in this study are expressed as mean ± S.D. The

experiments were performed in triplicate (n = 3). Analysis of variance (ANOVA)

followed by Dunnett’s post-test was used to determine the significant differences

between groups, and P value ≤ 0.05 and ≤ 0.01 were considered significant at 95% and

99% confidence. All statistical analyses were conducted using IBM SPSS (version 22)

for windows software.

3. Results

3.1 Effect of plaunotol on the cell viability

The MTT assay was performed to determine the cell viability of plaunotol in

human cancer cell lines. The growth of human cancer cell lines was inhibited when cells

were treated with plaunotol in a concentration-dependent manner. The relationship

between cell viability and concentration afforded the IC50 value. Table 2 summarizes

the IC50 of plaunotol and paclitaxel against the cell lines. It can be concluded that

plaunotol exhibits a moderate anti-proliferative activity.

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3.2 Effect of plaunotol on cell division

To understand the action of plaunotol on anti-proliferative activity, the effect of

plaunotol on cell division was performed by staining the DNA with propiodium iodide.

The cell cycle assay, using flow cytometer, indicated that the distribution of cell

population varied after treatment with plaunotol. The nuclear DNA intercalating stain

propiodium iodide discriminates cells at different stages of the cell cycle, based on

differential DNA content. Fig. 2 illustrates the histogram of cell population in different

types of cell lines. The results indicate that plaunotol affects cell division of HT-29 at

G2/M phase, and MCF-7 at S-phase. However, two types of cell lines like HeLa and

KB have slightly increased effect at G0/G1 phase with P-values of 0.036 and 0.01,

respectively, as shown in Fig. 3.

3.3 Effect of plaunotol on apoptosis

In treatment of the human cancer cell lines with plaunotol at 75 µM and 150

µM (equivalent to the IC50 and IC99, respectively) for 48 h, cells were prepared for

double staining according to the manufacturer’s protocol. After being subjected the

mixture to the MuseTM

analyzer, the population was gated. The results clearly showed

that plaunotol altered the membrane from apoptosis (Fig. 4A). This evidence appeared

in every cancer cell types, although with different sensitivity. As shown in Fig. 4B,

plaunotol triggered apoptosis in the early phase. It can be noted that high concentration

of plaunotol has cytotoxic effect in HT-29.

3.4 Plaunotol caused apoptosis in both extrinsic and intrinsic pathways

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After treating the human cancer cell lines with plaunotol (50 µM and 75 µM),

cells were harvested and the RNA extracted. The transcription profiles of apoptotic-

associated genes such as pro-apoptotic genes (TNF-α, BAX and BAK) and anti-apoptotic

genes (BCL-2) were determined when GAPDH was an endogenous gene. The relative

expression was calculated as the RQ value according to the mentioned equation in the

experiment.

In consideration of the expression profile of TNF-α, as shown in Fig. 5A,

treatment of cells with plaunotol increased the expression of TNF-α in HeLa and HT-

29. In contrast, TNF-α mRNA levels in MCF7 and KB cells were suppressed after

treating cells with plaunotol. For anti-apoptotic gene Bcl2, the expressions were

decreased in every cell type. On the other hand, plaunotol did not affect BAX and BAK

mRNAs in all cancer cell types. The ratio of expression level of BCL-2 and BAX was

estimated from the apoptotic-associate genes related to the apoptotic agent. The result in

Fig. 5B revealed that plaunotol caused apoptosis in HeLa, HT-29 and MCF7. In

conclusion, plaunotol play an important role in altering the cell division, causing

apoptosis by the suppression of apoptotic-associate genes via extrinsic and intrinsic

pathway in HeLa, HT-29 and MCF7, but not in KB cells.

4. Discussion

Plaunotol or (E,Z,E)-7-hydroxymethyl-3,11,15-trimethyl-2,6,10,14-

hexadecatetraen-1-ol has been registered with the World Health Organization (WHO),

under the name of CS-684 since 1983. It was manufactured in the form of soft-gelatin

capsule (combined with corn oil) under the tradename of KelnacTM

(Daiichi Sankyo

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Ltd., Tokyo, Japan). Since then, plaunotol has become an anti-peptic ulcer drug and is

recommended by physicians to be combined with antibiotics and proton pump inhibitors

for treatment of Helicobacter pylori-induced peptic ulcer (Takagi et al., 2000).

In the last three decades, pharmacological activities of plaunotol have been

investigated extensively with various publications. It possesses several pharmacological

activities, such as anti-inflammation, gastro-protection, antibacterial activity, and

anticancer properties. Plaunotol induces the prostaglandins production in gastric

mucosa, releasing endogenous secretin (Shiratori et al., 1993) and suppresses the

production of inflammatory mediators which are generated by leucocytes such as TNF-

α and IL-8 (Murakmi et al., 1999; Takagi et al., 2000). For anticancer activity, in

particular, only few reports on the effect of plaunotol in the cancer cells were found.

Plaunotol was reported to exert anti-cancer effect through its anti-angiogenic activity

(Kawai et al., 2005) and direct effect on gastric and colon cancer cells (Yamada et al.,

2007; Yoshikawa et al., 2009). Therefore, in the highlight of increasing value of C.

stellatopilosus, we proceeded to demonstrate the efficiency of plaunotol for anti-

proliferative and apoptosis activities.

The present study, anti-proliferative activity of plaunotol was reported against

four types of human cancers including human breast carcinoma cell line (MCF-7),

human cervical carcinoma cell line (KB), human cervix adenocarcinoma (HeLa), and

human colorectal adenocarcinoma (HT-29). In parallel, the human gingival fibroblast

was used represent a normal cell. Evaluation of cytotoxic activity with MTT assay

revealed that plaunotol exhibited dose-dependent anti-proliferative activity against all

human cancer cell lines and expressed an IC50 ranging from 60 µM to 80 µM. In

addition, at tested concentrations (< 100 µM), plaunotol was not toxic to normal cell

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line (HGF). In DLD1-human colon adenocarcinoma, plaunotol and geranylgeraniol

(GGOH) were shown to inhibit growth of the cancer cell by inducing caspase-mediated

apoptosis. These results showed that plaunotol not only inhibited growth of HT-29, a

human colon cancer cell, but also MCF-7, KB and HeLa cells.

In consideration of cell cycle arrest, several anti-cancer drugs are known for

inhibiting cancer growth and blocking cell cycle (Payne & Miles, 2008), like paclitaxel,

for instance. The cell cycle is the control process in eukaryotic cell, which evaluate the

condition of the genetic formation during cell division. Its mechanism is regulated by

three internal checkpoints including G0/G1 phase, S phase, and G2/M phase. G1

checkpoint is a major checkpoint caused by damage of DNA and the cell cannot pass to

the next stage (S phase). S checkpoint checks the replication of DNA prior to

undergoing the mitosis stage. The last stage, M checkpoint, evaluates whether all sister

chromatids are correctly attached to spindle microtubules before the cell enters the

irreversible anaphase. From the results, the human cancer cell lines have different

response to plaunotol at 75 and 100 µM. Plaunotol exhibited anti-proliferative activity

at the resting stage (G0/G1) of HeLa and KB. On the other hand, it caused an inhibition

during DNA synthesis (S) of MCF-7 and during cell division (G2/M) of HT-29. It can

be concluded that plaunotol has an anti-proliferative activity, affecting the cell division

at different stages depending upon the type of cancer cell.

The inhibitory effect of plaunotol against HeLa, HT-29, MCF-7 and KB-cells

was confirmed by staining the cells with Annexin V. The result indicated that HeLa,

HT-29 and MCF-7 were sensitive to plaunotol at 75 and 150 µM and induced at early

stage of apoptosis. In contrast, plaunotol had less effect on KB-cells, which is a

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derivative of HeLa cells. A different mechanism of plaunotol on anti-proliferative

activity in the KB-cells can be postulated.

Plaunotol has been reported to induce apoptosis of gastric cancer cells

(Yamada et al., 2007). Moreover, it activated caspases and induced apoptosis in colon

cancer (Yoshikawa et al., 2009). The present study clearly showed that plaunotol acted

as an apoptotic agent in breast cancer, as well as cervix and colon cancers. It blocked

the cell cycle during cell division, altered membrane of the cells causing cell death and

inhibited the BCL-2 expression, resulting in apoptosis of the cancer cells. In summary,

plaunotol induces apoptosis through death receptor and mitochondrial dependent

pathway. Altogether, the present study provided supportive data on the anti-proliferative

activity and apoptotic mechanism of plaunotol in human cancer cells. Thus, plaunotol

may have a therapeutic potential or exert on chemotherapy for the treatment of human

cancer.

Acknowledgements

This work was funded from the Annual Government Statement of Expenditure,

Prince of Songkla University (PSU); under Grant number PHA580237S; the PSU

Graduate School; and the Thailand Research Fund through the Golden Jubilee PHD

program under Grant number PHD/071/2553)

References

Chaotham, C., Chivapat, S., Chaikitwattana, A., & De-Eknamkul, W. (2013). Acute and

chronic oral toxicity of a partially purified plaunotol extract from Croton

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10.1155/2013/303162

Cooper, G.M. (2000). The cell: a molecular approach. Washington, D.C: ASM Press.

Fuchs, Y. & Steller, H. (2011). Programed cell death in animal development and

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Figure caption

Figure 1. Chemical structure of plaunotol.

Figure 2. Histograms of DNA content profiles in the HeLa, HT-29, MCF7 and KB cells after

treatment at 75 µM and 100 µM plaunotol in comparison with control and paclitaxel

treatments for 48 h. Cells were stained with propiodium iodide and analyzed by flow

cytometry.

Figure 3. Percent of cell population of human cancer cell lines after treatment with different

concentrations of plaunotol. Data were expressed as mean ± S.D of triplicate

experiments and was analyzed by ANOVA followed by Dunnett’s post-test where **

was at P < 0.001 and * was at P < 0.05 when compared to control.

Figure 4A. Dot plots indicate amount of stained cells in each quadrant: live, early apoptosis, live

cells, late apoptosis and dead cells, respectively in HeLa, HT-29, MCF7 and KB cells.

Percentage of each quadrant indicates the population of cells obtained from gating.

Figure 4B. Summary of the percent population of cells after treatment with plaunotol at 75 µM

and 150 µM in different type of cancer cells in comparison with control (0.2%

DMSO). The experiment was performed in triplicate. Data were analyzed by ANOVA

followed by Dunnett’s post-test (*, ** indicate P value < 0.05 and < 0.001,

respectively).

Figure 5. The relative expression levels of apoptotic-associate genes in the human cancer cell

lines (HeLa, HT-29, MCF-7 and KB cells) after treated with plaunotol at 50 µM and

75 µM for 48 h. (A) the RQ values of TNF-α, BCL-2, BAX and BAK. (B) the RQ

ratios of RQBCL-2 and RQBAX.

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Figure 1

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Figure 2

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* *

*

0

20

40

60

80

100

G0/G1 S G2/M

% of cell population

Control 25 µM 50 µM 75 µM 100 µM

*

**

**

0

20

40

60

80

100

G0/G1 S G2/M



**

**

0

20

40

60

80

100

G0/G1 S G2/M



*

**

**

***

0

20

40

60

80

100

G0/G1 S G2/M



HeLa HT-29

MCF7 KB

Figure 3

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Figure 4A

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Figure 4B

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Bak

Bak

Bak

Bak

TNF-α BCL-2 BAX BAK

Figure 5

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RQBCL-2 / RQBAX

RQBCL-2 / RQBAX

RQBCL-2 / RQBAX

RQBCL-2 / RQBAX

Figure 5

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Table 1. Primers used in qRT-PCR experiment.

Name Accession number*

(location)

Sequence (5′→3′) Product size

(bp)

F-TNFα

R-TNFα

NM_000594.3

(263-463)

TGC TTG TTC CTC AGC CTC TTC TC

AGG GTT TGC TAC AAC ATG GGC T

200

F-BCL2

R-BCL2

NM_000633.2

(1015-1144)

CCT GTG GAT GAC TGA GTA CCT GTG

CAC AG AGA CAG CCA GGA GAA ATC A

129

F-BAX

R-BAX

NM_001291428.1

(335-440)

GAG AGG TCT TTT TCC GAG TGG C

GCC TTG AGC ACC AGT TTG CTG

105

F-BAK

R-BAK

NM_001188.3

(1366-1525)

GAG AGG TCT TTT TCC GAG TGG C

GCC TTG AGC ACC AGT TTG CTG

159

F-GAPDH

R-GAPDH

NM_001256799.2

(1066-1245)

ACC CAC TCC TCC ACC TTT GAC

TCC TCT TGT GCT CTT GCT GG

179

* Retrieved from https://www.ncbi.nlm.nih.gov/

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Table 2. Anti-proliferative activity of plaunotol against the human cancer cell lines (n=3).

Compound

(conc.)

IC50 in different cell line*

HeLa HT-29 MCF-7 KB

Plaunotol (µM) 65.5±6.4 72.9±5.7 62.3±9.1 80.9±3.5

Paclitaxel (nM) 12.3±2.9 7.6±4.0 4.5±2.5 11.4±0.5

*The IC50 on the human gingival fibroblast was > 100 µM.

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